The capacity of cells to migrate over surfaces plays a key role in embryonic development, wound healing, defense against microbial infection, homing to target organs, and metastasis of tumors. The spectrum of cells that display this type of locomotion encompasses a broad range of phenotypes but each is thought to be propelled by cytoplasmic contraction of actin-myosin complexes. The amoeboid spermatozoa of nematodes are an exception. These cells exhibit the same motile behavior as other amoeboid cells but lack both actin filaments and myosin. Their pseudopods, instead, contain arrays of filaments, composed of a 15,500 protein, that exhibit patterns of organization and movement consistent with a central role in sperm locomotion. The purpose of this proposal is to analyze this new motility system in detail using sperm from two species of nematodes, Caenorhabditis elegans and Ascaris lumbricoides. Filaments will be isolated from sperm for analysis of their structure and organization by high resolution electron microscopy. The capacity of these filaments to self-assemble in vitro will be exploited to define the chemical requirements and kinetics of their polymerization. In order to assess the role of the filaments in locomotion, computer-enhanced video microscopy will be used for a stepwise dissection of motility, starting with intact sperm and proceeding to isolated filament complexes. In addition, accessory proteins involved in organizing the filaments into three- dimensional arrays, linking these arrays to the plasma membrane, and generating force within the filament system will be identified and isolated and their specific interactions with filaments defined by in vitro assays. The long-term objective of this work is to understand the machinery of sperm locomotion in molecular detail and determine how this new system of motility relates to other types of cellular motors.